Modern genetic engineering offers tremendous opportunities to develop biotech organisms with commercially desirable characteristics or traits. Particularly, recent advances in molecular biology and transgenic technologies have greatly accelerated the introduction of new genes and, hence new traits into commercial crops. The proper expression of a desirable transgene in a transgenic organism is widely considered to be a requisite requirement to achieve this goal. Nucleic acid elements having gene regulatory activity, i.e. regulatory elements such as promoters, leaders, enhancers, introns and transcription termination regions, are polynucleotide molecules which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in a crop of interest are therefore useful for modifying the crop's characteristics through the methods of genetic engineering.
In the field of algae biotechnology, transgenesis of algae is a complex and fast growing technology. A powerful driving force in algal transgenesis is the promising prospect of using genetically modified algae as bioreactors. In fact, non-transgenic algal biotechnology has been deployed in many technology areas including nutrition, aquaculture, production of chemicals and pharmaceuticals, etc. In particular, non-transgenic microalgae have proven their utility and tractability as a production system for therapeutic or industrial products and, in this respect, algae now seem poised to become the “green” alternative to current mammalian, yeast, insect, or even bacterial recombinant production systems. Furthermore, recent progress in algal transgenesis promises a much broader field of application in molecular farming, which is generally defined as the production of proteins or metabolites that are valuable to medicine or industry, and has become increasingly feasible with transgenic algal systems. Indeed, the ability of transgenic algae to produce high levels of recombinant antibodies, vaccines, insecticidal proteins, or bio-hydrogen has already been demonstrated in several microalgal species.
As a result, there is a continuing need for novel genetic tools and methods that would facilitate the genetic engineering of algae to further enhance their physiological properties. In particular, several microalgae have recently attracted considerable attention as being potentially suitable for algal biofuel production. However, optimization of culture conditions for selected microalgal species has been reported to be potentially a challenge, because the fatty add content of individual species and isolates can vary considerably under different environmental conditions in laboratory culture and in large-scale production field. For these reasons and others, it is of immense social, ecological and economic interests to develop novel algal strains that have enhanced nutritional value, improved resistance to biotic contaminations, and tolerance to harsh conditions such as high salinity and high temperature. Therefore, more efficient methods and systems for large-scale cultivation of microalgae are critical if algal-derived biofuels are to become a reality. If these issues can be resolved, algae will potentially represent a far more superior source of biofuel than terrestrial plants. Optimization of biofuel production in algal systems should further improve the potential of this auspicious technology in the future.
However, despite the availability of many molecular tools, the genetic modification of algae, particularly microalgae, is often constrained by an insufficient expression level or temporally nonspecific expression of the engineered transgene. In addition, while previous work has provided a number of regulatory elements that can be used to affect gene expression in transgenic algae, there is still a great need for novel regulatory elements with beneficial expression characteristics. One example of this is the need for regulatory elements capable of driving gene expression preferentially in different algal growth phases. On the other hand, there exists a need for regulatory elements capable of driving gene expression constitutively throughout cell life cycle and/or unaffected by growth conditions. Thus, the identification of novel molecular tools including genes, vectors, regulatory elements (e.g., promoters), etc. that function in various types of algae and in distinct growth phases and growth conditions will be useful in developing enhanced varieties of algae.
Furthermore, as the field of algal transgenesis develops and more genes become accessible, a greater need exists for algae transformed with multiple genes. These multiple exogenous genes typically need to be transcriptionally controlled by separate regulatory sequences. For example, some transgenes need to be expressed in a constitutive manner whereas other genes should be expressed at certain developmental stages or in specific compartments of the transgenic cell. In addition, multiple regulatory sequences are also needed in order to avoid undesirable molecular interactions which can result from using the same regulatory sequence to control more than one transgene.